To evaluate the ability of Porphyromonas gingivalis' cysteine proteases (gingipains) to cleave the innate immune receptors involved in bacterial recognition and to evaluate the consequences of cleavage for the cellular response to infection with live bacteria.
Methods:
Primary murine peritoneal macrophages or macrophage cell lines were exposed to supernatants of wild type, Rgp- or Kgp- P.gingivalis or to purified gingipains (RgpA, RgpB or Kgp) for 5-60 minutes. Macrophage surface expression of TLR2, TLR4 and CD14 was determined by flow cytometry. TNF-α secretion by CD14 cleaved RAW246.7 macrophages or peritoneal elicited macrophages (1uM Kgp, 30-minutes) or CD14-/- macrophages was measured (ELISA) following stimulation with increasing MOI of P. gingivalis.
Results:
RgpA and Kgp decreased CD14-detection in a concentration (P=0.0000002) and time (P=0.03) dependent-manner, whereas RgpB did not have a significant effect. The type of gingipain influenced the strength of CD14-cleavage with Kgp>>RgpA>>RgpB. Kgp-mediated CD14 cleavage appeared faster and with lower protease concentration than with RgpA. Gingipains did not significantly affect TLR2 or TLR4 expression on murine-macrophages.
Macrophage TNF-α secretion following stimulation with live P.ginginvalis (MOI 10,50) was significantly reduced by pre-treatment with Kgp (p value<0.05). Furhermore, TNF-α secretion by CD14-/- macrophages following live P.gingivalis stimulation was significantly lower compared to RAW-macrophages (p value<0.01 for MOI 10,25; p value<0.05 for MOI 50,100).
Conclusions:
The gingipain hemaglutinin/adhesion site is important in cleaving murine CD14 since only RgpA and Kgp efficiently cleaved it. This cleavage resulted in decreased macrophage TNF-α secretion following live P. gingivalis challenge. The fact that gingipains demonstrate selective cleavage of key innate-immune receptors leading to decreased TNF-α levels suggests that they play an important role in modulating the host immune response to P. gingivalis, setting the stage for chronic infection.